Induction of immunological tolerance

ABSTRACT

Immunosuppressive agents which are conjugates of an antigen linked to a D glutamic acid:D-lysine copolymer are disclosed. Also disclosed are methods of preparing the conjugates and therapeutic methods for inducing immunological tolerance to antigens.

This is a division of Ser. No. 764,586, filed Feb. 3, 1977.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to conjugates suitable for inducing in anindividual immunological tolerance to an antigen. The conjugatesfunction by suppressing the formation of antibodies to a specificantigen. Antigens can be defined as macromolecules which will, whenintroduced into an individual, cause the production of antibodies bythat individual and will react specifically with those antibodies.

The basic function of the organs, cells and molecules that comprise theimmune system is to recognize and to eliminate from the body foreignsubstances. These foreign substances are eliminated by reaction betweenthe foreign substance and antibodies which are formed in response to thesubstance. In general, this function is performed efficiently andwithout detriment to the host. However, in certain instances,disturbances can occur which can lead to pathogenic disorders such as,for example, an uncontrolled response (allergic disorders) or anabnormal response (autoimmune disease). The pathogenesis of both thesedisorders is related directly or indirectly to the production ofantibodies to either environmental antigens (allergens) orself-antigens. In addition, since the function of the immune system isto recognize and eliminate foreign substances, transplantation ofhealthy tissue and organs from a donor to a genetically non-identical(i.e., allogenic) acceptor individual is difficult to achieve because ofthe allograft reaction.

If an individual undergoes an "altered state" as a result of contactwith an antigen (and formation of antibodies thereto), subsequentcontact with that antigen or a structurally similar substance can evokea pathological reaction. Such individuals are termed "hypersensitive",with regard to one or more specific reaction-provoking antigens. Whenthese individuals inhale or ingest the appropriate antigen, a prominentand common manifestation includes hayfever, asthma or hives. Thetendency to develop this form of allergy ("atopy") is hereditable.

An individuals first antibody response to an antigen evokes a smallerand somewhat different antibody response than the response evoked uponsubsequent exposure. The first exposure to an antigen evokes a primaryresponse. After antibody levels in the primary response have declined,even to the point of no longer being detectable, a subsequent encounterwith the same antigen usually evokes an enhanced secondary (anamnestic)response.

The appearance of this atopy is involved with the production within anindividual of a type of tissue-sensitizing IgE antibody called a reagin.These IgE antibodies have a high affinity for receptors on cells presentin various body tissues. The receptors are on mast cells which are foundin close association with capillaries in connective tissues throughoutthe body and on basophilic leukocytes (blood cells). Mast cells andbasophils contain a high content of pharmacologically-active mediators,such as histamine, serotonin, (5-hydroxytryptamine) and kinins (basicpeptides), concentrated in cytoplasmic granules. Contact of the IgEantibodies, which are fixed to mast cells and basophils, with antigenscan trigger cross-linking of the IgE antibodies. In turn, thiscross-linking causes degranulation of mast cells and basophils, whichreleases the chemical mediators and produces manifestations of theallergic response referred to earlier.

While the appearance of atopy is dependent upon production of cell-bound(IgE) antibodies, another type of antibodies of importance to the immunesystem is of the IgG class. These IgG antibodies are referred to ascirculating antibodies or "blocking" antibodies. IgG antibodies are alsocapable of combining with antigens. This combination can inactivateantigens by "blocking" the ability of the antigen to react withcell-bound IgE, and subsequent cross-linking of the IgE antibodies.

A common method of treating allergic disorders is by immunizing("desensitizing") the individual by repeated injections of small,increasing amounts of antigen, at intervals, eg., weekly, and at dosagelevels that avoid triggering of the degranulation of mast cells orbasophils. It is believed that repeated injections increase the level ofblocking IgG antibodies, but not the level of cell-bound IgE antibodies.

This desensitizing approach is subject to a number of disadvantages.Therapeutic benefits are difficult to achieve consistently, and thetreatment is tedious. In addition, since exposure to the environmentalantigen causes subsequent production of IgE antibodies, the possibilityof an IgE-antigen reaction and subsequent IgE cross-linking, is alwayspresent.

An autoimmune disease is a pathological condition arising from anautoimmune response in which an individual responds immunologically byproduction of antibodies to a self-antigen. Autoimmunity can affectalmost every part of the body, and generally involves a reaction betweena self-antigen and IgG antibody. Representative autoimmune diseases caninvolve the thyroid, gastric mucosa, adrenals, skin, red cells andsynovial membranes.

For some types of autoimmune diseases, non-specific immunosuppressanttreatment, such as whole body X-irradiation or the administration ofcytotoxic drugs, has been used with limited success. The disadvantagesof such treatment include the toxicity of the agents used, and theincreased incidence of various cancers, especially lymphomas andreticulum cell sarcomas, following such therapy. In addition, the use ofnonspecific agents for chronic immunosuppression greatly increases thesusceptibility of the patient to serious infection from environmentalfungi, bacteria and viruses which under ordinary circumstances would notcause problems. The invention disclosed herein is specific and onlysuppresses the antibody response to the offending antigen.

In contrast to the "blocking" desensitization approach of treatment ofenvironmental allergies with antigen extracts, and the non-specificimmunosuppression of autoimmune diseases, the present invention providesa means of inducing a long-lasting state of specific immunologicaltolerance by suppression of formation of antibodies to specificantigens.

2. Prior Art

In viewing the prior art in the field of immunology, it is important torecognize the distinction between an antigen and a hapten. As definedhereinbefore, antigens cause the production of antibodies and also reactspecifically with the antibody produced. In contrast, haptens aredefined as a small molecule which by itself cannot stimulate antibodyproduction, but will combine with an antibody, once formed. Further,haptens as a rule do not induce cellular immunity, cannot serve ascarriers for other haptens and induce antibody formation only whenintroduced on immunogenic carriers. An anti-hapten antibody response hasstrict carrier specificity. A secondary response to the hapten can onlybe elicited when it is administered on the same carrier; if it isintroduced on an immunologically unrelated carrier, an individual willshown no immunological memory for the hapten and will give a typicalprimary response.

Because the capacity for a secondary response can persist for many yearsit can provide a long-lasting immunity. The primary response is lessprotective, because antibodies appear more slowly. In a series of papersone of the inventors and his co-workers demonstrated and characterized asystem of prolonged hapten-specific bone-marrow derived cell toleranceusing the D-glutamic acid:D-lysine copolymer to which the appropriatehapten had been attached. [See J. Exp. Med., Vol. 134, pp. 201-223(1971); Vol. 136, pp. 1404-1429 and pp. 426-438 (1972); Vol. 138, pp.312-317 (1973); Vol. 139, pp. 1446-1463 (1974) and Proc. Natl Acad. Sci.U.S.A. Vol. 71, pp. 3111-3114].

These studies demonstrated success in inducing tolerance in thebone-marrow derived lymphocyte precursors of antibody forming cells ofantibody classes which were specific for the 2,4 dinitrophenyl (Dnp)hapten. The investigations involved the use of conjugates of Dnp andD-glutamic acid:D-lysine, (hereinafter D--GL).

Recent studies by one of the inventors and co-workers demonstrated thattolerance to nucleoside determinants could be obtained by usingconjugates of nucleosides and D--GL. [See J. Immunol., Vol. 114, pp.872-876 (1975)]. The nucleoside work dealt with the induction oftolerance to a mixture of nucleosides, which are made up of aheterocyclic base and a five-carbon sugar. This work is similar toinduction of tolerance to DNP--D--GL.

While these immunological studies are of interest in demonstratingsuppression of antibody response to chemical moieties which function assingle determinants when coupled to appropriate non-immunogenic carrierssuch as D--GL copolymer, no immunotherapeutic application to antigens isdisclosed.

The experimental results herein which relate to penicillin allergy, andthe use of the major antigenic determinant, benzylpenicilloyl (BPO), aredescribed in Proc. Natl. Acad. Sci. U.S.A., Vo. 73, No. 6. pp. 2091-2095(1976).

SUMMARY

The subject matter of the present invention includes:

(a) a therapeutic immunosuppressive agent capable of suppressingspecific antibody formation in an individual. The agent is a conjugateof D-glutamic:D-lysine copolymer and an antigen. The antigen can beeither an allergen or a self-antigen. Allergens include:benzylpenicilloyl, insulin, ovalbumin, lactalbumin, bermuda grasspollen, timothy grass pollen, orchard grass pollen, and combinations ofgrass pollens, ragweed pollen, ragweed antigen E, birch tree pollen, beevenom, snake venom, horse dander, cat epithelial, haddock, house dustmite, Chrysanthemum leucanthemum, Alternaria tenuis, trypsin,chymotrypsin, dry rot, baker's yeast, tetanus toxoid, diphtheria toxin,ficin and derivatives thereof. Self-antigens include: nucleic acid,oligodeoxynucleotide, thyroglobulin, thyroid cell surface or cytoplasm,parietal cell, adrenal cell, epidermal cell, uvea cell, basementmembrane cell, red cell surface, platelet cell surface, muscle cell,thymus myoid cell, mitochondria, secretory duct cell, deoxyribonucleicacid-protein, acetylcholine receptor substance, insulin and other normalhormone and tissue factors.

(b) a method for preparing a therapeutic immunosuppressive agent byreacting a D-glutamic acid:D-lysine copolymer with an antigen. Thecoupled antigen-D-GL conjugate is recovered by conventional purificationtechniques; and

(c) a therapeutic method of inducing immunological tolerance to anantigen in an individual for whom such therapy is indicated, byadministration of a conjugate of antigen and D-glutamic:D-lysinecopolymer, as defined above.

DESCRIPTION OF THE INVENTION

The present invention relates to induction of immunological tolerance tospecific offending antigens. The antigen is coupled to a D--GLcopolymer. The immunological tolerance achieved can be defined as aspecific unresponsive state in which the individual fails to formantibodies, in response to the introduction into the individual, of theantigen. The tolerance induced was manifested by:

(1) the inability of an individual treated with antigen-D-GL to developprimary antigen-specific antibody responses;

(2) the ability of antigen-D-GL conjugate to abrogate an ongoinganti-antigen antibody response; and

(3) the inability of an individual previously primed with an antigen togenerate secondary anti-antigen responses following treatment withantigen-D-GL conjugate.

The suppression is accomplished by administering to that individual anamount of the antigen-D-GL conjugate that has an effective suppressingeffect upon specific antibody response. The term "individual" asutilized in this specification means a human being or an experimentalanimal that is a model for a human being. Medical indications for theuse of the conjugate of the present invention are any conditions inwhich it is desired to suppress within the individual antibody responseto a specific antigen. The term "suppression of antibody response" orany equivalent of that term means a significant increase inimmunological tolerance to a specific antigen. This suppression isaccomplished by administering to the individual a dose, or series ofdoses, which will suppress or decrease antibody response. Although theamount will vary from individual to individual and from indication toindication, it is easily determined by one skilled in the art withoutundue experimentation. Subcutaneous administration is preferred. Doseforms for administration of the conjugate can be prepared by recognizedmethods in the pharmaceutical sciences.

Hypersensitivity reactions to drugs, such as penicillin, are a commonallergic disorder in humans. The mechanisms involved which areillustrated with penicillin can be considered as models for allergies ingeneral. In order to conduct a thorough investigation of suppression ofspecific antigen-antibody response, a penicillin model of allergy wasused.

Penicillin is relatively unstable and most of its solutions contain atleast small amounts of penicillinate, a highly reactive derivative thatforms penicilloyl and other substituents of amino and sulfhydral groupsof proteins. Penicillin G, derived from crystalline potassiumbenzylpenicillin G (KPB), the most widely used pencillin, has a benzylgroup attached to the carboxyl group; the major antigenic determinant ofpenicillin G, that is, the restricted portion of the antigen moleculewhich determines the specificity of the antibody-antigen reaction, isbenzylpenicilloyl (hereinafter BPO).

The process for producing the antigen D--GL conjugates of the presentinvention involves dissolving D--GL copolymer in an alkaline solutionand reacting the alkaline solution with from about 2 to 3 molarequivalents of the antigen. The reaction mixture is maintained at atemperature of from about 10° to 30° C. for about one hour. The pH ofthe reaction mixture is maintained in a range of about 10-12, byaddition of a alkaline material e.g., KOH or NaOH. The antigenconjugates are washed and purified by known techniques, e.g. dialysis.Suitable copolymers, having a molecular weight of about 34,000 about50,000 and about 64,000, respectively, and a glutamic acid:lysine molarratio of 60:40 are available from Miles Laboratories, Inc., 1127 MyrtleStreet, Elkhart, Ind., 46514.

In order to determine the immunospecific characteristics of theconjugates of the present invention, high-titered IgE, IgG and IgMantibody responses to an antigen were elicited in mice by theintraperitoneal (i.p.) injection of an antigen keyhole limpet hemocyanin(KLH). The amount of antibody produced in response was then measured bythe assay techniques described hereinafter. Treatment of such mice withthe antigen-D-GL, according to the present invention, either before orafter the primary immunization, resulted in significant suppression ofthe subsequent anti-antigen antibody response of the IgE and IgGclasses, measured at both the humoral and cellular level.

ASSAY TECHNIQUES I. BPO-Carrier Conjugates

(a) BPO--KLH and BPO--BSA

BPO was coupled to keyhole limpet hemocyanin (KLH) and to bovine serumalbumin (BSA) as described in J. Clin. Invest. 47, pp. 556-567 (1968)and Int. Arch. Allergy, 39, pp. 156-171 (1970). The proteinconcentration of the conjugates was determined by Kjeldahl nitrogenanalysis (with a correction for the amount of nitrogen contributed bythe BPO groups). The conjugates were assayed for BPO content bydetermining the penamaldate concentration. The penamaldate measurementinvolves spectrophotometric quantitative determination of the BPO-D-GLconjugate. [See Methods in Immunology and Immunochemistry, AcademicPress, pp. 141-142 (1967)]. The molar ratio of BPO/KLH and BPO/BSAobtained was:

BPO₃₅ --BSA (10 equivalents of potassium benzylpenicillin equivalent to.sub.Σ --NH₂);

BPO₁₀ --KLH (10 equivalents of potassium benzylpenicillin equivalent to.sub.Σ --NH₂ ; molecular weight of subunit of KLH of 100,000, withestimated 50.sub.Σ --NH₂ groups).

(b) BPO-SRBC (Sheep Erythrocytes)

For use in testing serum BPO-specific IgM and IgG antibodies, BPO wascoupled to SRBC by the method described in J. Immunol. 96, pp. 707-718(1966).

The BPO-carrier conjugates, prepared as described above were utilized inimmunization of mice or in measurement of anti-BPO antibodies asdescribed in detail hereinafter.

II. Immunization Procedure

Mice were immunized by intraperitoneal (i.p.) injection of 1 μg ofBPO--KLH adsorbed on 4 mg of Al(OH)₃ gel (alum) in a volume of 0.5 mlsterile saline. Booster injections were given i.p. from 2 to 4 weeksafter primary injections. Booster injections were made with 1 μgBPO--KLH mixed with 2 mg of alum.

At various intervals after primary and secondary immunization, mice werebled from the retro-orbital plexus and the serum antibody levelsdetermined as described below.

III. Measurement of Anti-BPO Antibodies

Serum IgE antibodies.

Passive Cutaneous Anaphylaxis (PCA)

The PCA method involves pooling sera from a given group of mice andserially diluting (2-fold) the sera in 2 percent normal rat serum. A 0.1ml portion of each of the various dilutions was injected intradermallyinto the shaven dorsal skin of the test rats. After a 4-24 hoursensitization period, the PCA reactions, which measure only IgEantibodies by this technique, were elicited by intravenous (i.v.)injection of BPO--BSA (1 mg/250 gm of shaved rat body wt) in 1.0 percentEvans' blue dye dissolved in phosphate-buffered saline. The PCA titer isexpressed as the reciprocal of the highest dilution of serum yielding a5 mm diameter blueing reaction. [See Life Sciences, 81, pp. 813-820(1969)].

IV. Measurement of Serum Anti-KLH Antibodies

Serum IgE anti-KLH antibody levels were determined by PCA reactions asdescribed above. IgG anti-KLH antibodies were determined byradioimmunoassay using ¹²⁵ I-labelled monomeric KLH as described in J.Immunol. 114, pp. 872-876 (1975).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following examples illustrate preparation of the conjugates of thepresent invention.

EXAMPLE I BPO-D-GL Conjugate

A one gram portion of D-GL copolymer having an average molecular weightof about 50,000, and a glutamic acid:lysine residue molar ratio of60:40, was dissolved in 0.1 m sodium carbonate solution (pH=11.5). ThepH was maintained at about 10-12 by additions of 1 NaOH. From 2 to 3molar equivalents of potassium benzylpenicilloyl was added, and thereaction mixture maintained at a temperature of about 10° to 30° C. forabout 1 hour.

The resultant BPO--D--GL conjugate was separated from the unreactedpenicillin salt by dialysis purification. The dialysis involved severalchanges of 0.1 M sodium bicarbonate containing 1 percentdiethylaminoethyl cellulose (DEAE), and finally againstphosphate-buffered saline.

Analysis of the BPO--D--GL conjugate by the method referred to earlierindicated that the BPO--D--GL molar ratio was BPO₄₀ --D--GL (2equivalents potassium benzylpenicillin/equivalent .sub.Σ --NH₂). TheBPO--D--GL conjugate formed has a molar ratio of benzylpenicilloyl, or aderivative thereof, to copolymer, of at least 40:1.

High titered IgE, IgG and IgM antibody responses to BPO were elicited bythe i.p. injection of BPO--KLH. Treatment of such mice, as describedhereinafter, with the BPO--D--GL according to the present invention,either before or after the primary immunization, resulted in significantsuppression of the subsequent anti-BPO antibody response of the IgE andIgG classes, measured at both the humoral and cellular level.

Induction of BPO-Specific Tolerance With BPO--D--GL When BPO--D--GLTreatment Precedes Primary Immunization.

Analysis of Humoral Immune Responses.

Two groups of normal BALB/c mice were injected subcutaneously (s.c.)with four doses of either saline or 500 μg of BPO--D--GL at 3 dayintervals. This regimen was chosen on the basis of preliminaryexperiments which demonstrated that: (1) the subcutaneous route is asgood as or better than intraperitoneal for tolerance induction withD--GL conjugates, and (2) two 500 μg doses of BPO--D--GL resulted in asignificant, but incomplete degree of tolerance. One week after the lastdose, animals were primarily immunized with 1 μg of the sensitizingantigen BPO--KLH mixed with 4 mg of alum, an immunization procedurewhich was found to induce good IgE, IgM and IgG primary anti-BPOantibody responses in such mice. All animals were bled at weeklyintervals and their sera analyzed for anti-BPO and anti-KLH antibodies.On day 28 after primary immunization, the mice in both groups weresecondarily challenged with 1 μg of BPO-KLH mixed with 2 mg of alum; 7days later they were bled and sacrificed. The protocol and data of serumantibody responses are summarized in Table 1. Control animals developedgood primary IgE anti-BPO antibody responses by day 14, which peaked byday 21; these animals manifested sharp anamnestic responses on day 35following secondary challenge on day 28. In contrast, mice which hadbeen pretreated with BPO-D-GL failed to produce detectable IgE anti-BPOresponses over the 28 day primary course and produced only low amountsof antibody following secondary challenge. Comparable IgE anti-KLHantibody titers between treated and control mice over the 35 dayobservation period indicated tolerance specificity.

Serum BPO-specific IgM and IgG antibodies were determined by passivehemagglutination utilizing BPO coupled to sheep erythrocytes asdescribed in J. Immunol. 111, pp. 638-640 (1973). The anti-BPO antibodyresponses of the IgG and IgM class were similar to the IgE class resultsdescribed above. Mice treated with BPO--D--GL displayed significantlylower levels of IgG anti-BPO serum antibody over the entire immunizationperiod and following secondary challenge as compared to controls.

Spleen cells were removed under sterile conditions, and analyzed forBPO-specific plaque forming cells by the technique described in Science140, pp. 405-411 (1963). Heterologous adoptive cutaneous anaphylaxisreactions were performed as described in J. Immunol. 111, pp. 638-640(1973). The data indicated that substantially fewer BPO-specificantibodies of the IgG and IgM classes were present in spleen cells ofmice treated with BPO--D--GL as compared with untreated control mice.

                  TABLE I                                                         ______________________________________                                        Sera Antibody Response                                                        Effect of BPO-D-GL Pretreatment on                                            Primary IgE Anti-BPO Antibody Responses                                       of BALB/c Mice to BPO-KLH (PCA Titer)                                         PROTOCOL             SERUM ANTI-BPO                                                              Days After                                                                              ANTIBODY                                         Group Pretreatment Priming   IgE                                              ______________________________________                                                            7        <10                                                    (Control)    14        160                                              I     Saline s.c.  21        320                                                                 28        320                                                                 35        2560                                                                 7        <10                                                                 14        <10                                              II    BPO-D-GL s.c.                                                                              21        <10                                                    (500 μg × 4)                                                                      28        <10                                                                 35        40                                               ______________________________________                                         Secondary challenge was administered on day 28.                          

Induction of BPO-Specific Tolerance in Previously Immunized Mice byAdministration of BPO--D--GL.

Three groups of BALB/c mice were immunized i.p. with 1 μg of BPO--KLHmixed with 4 mg of alum. Two weeks later, the groups were bled and thentreated with either saline, 500 μg of BPO--D--GL i.p. or 500 μg ofBPO--D--GL s.c. on two alternating days (days 14 and 16). On day 18, allanimals were secondarily challenged with 1 μg of BPO--KLH mixed with 2mg of alum and bled at 7 day intervals over the ensuing 3 weeks.Twenty-one days after secondary immunization, the animals weresacrificed and their spleen cells assayed for BPO-specific plaqueforming cells.

The protocol and data of serum antibody responses are summarized inTable II. All 3 groups of mice displayed comparable levels of IgEanti-BPO antibodies immediately preceding BPO--D--GL treatment.Following secondary challenge with BPO--KLH, the untreated control micemanifested anamnestic IgE responses which plateaued on days 7 and 14 anddeclined somewhat by day 21 after challenge. In contrast, the 2 groupstreated with BPO--D--GL failed to respond to secondary immunization withBPO--KLH and, moreover, exhibited a decrease in circulating IgE anti-BPOantibody levels, this being most marked in the group treated withBPO--D--GL subcutaneously; the IgE titer in the latter group declinedprogressively until none was detectable by day 21. Comparable IgEanti-KLH antibody titers between treated and control mice over the 21day observation period indicated tolerance specificity.

Similar findings in anti-BPO antibody responses of the IgG class wereobtained. Thus, mice treated with BPO--D--GL were substantiallysuppressed in their IgG anti-BPO responses as compared to controls.

                  TABLE II                                                        ______________________________________                                        Effect of Intervening Treatment with BPO-D-GL                                 On Secondary IgE Anti-BPO and Anti-KLH                                        Antibody Responses of BALB/c Mice to BPO-KLH                                  PROTOCOL             SERUM ANTI-BPO                                                             Days After ANTIBODY                                               Intervening Secondary  (PCA TITER)                                      Group Treatment   Challenge  IgE                                              ______________________________________                                                          -4         320                                              I     (Control)   7          1280                                                   Saline      14         1280                                                               21         300                                                                -4         320                                              II    BPO-D-GL i.p.                                                                             7          40                                                     (500 μg × 2)                                                                     14         40                                                                 21         40                                                                 -4         320                                              III   BPO-D-GL s.c.                                                                             7          40                                                     (500 μg × 2)                                                                     14         20                                                                 21         <10                                              ______________________________________                                    

Spleen cell tests indicated that the level of BPO-specific antibodies ofthe IgG and IgM class in the spleens of treated mice were lower than inthe spleens of untreated controls.

From the above, it can be seen that the present invention provides amethod whereby a state of immunological tolerance to a specific antigencan be induced in an individual by administration of the appropriateantigen-D-GL conjugate. The tolerance is manifested in the IgE as wellas the IgG and IgM antibody class. In addition, the test results showthat tolerance can be established irrespective of the immune status ofthe animal at the time of treatment.

EXAMPLE II BPO--D--GL Conjugate

A one g portion of D--GL copolymer, having an average molecular weightof about 64,000 and a glutamic acid:lysine residue molar ratio of 60:40,and a 0.5 g portion of potassium benzylpenicilloyl were dissolved in 0.1M sodium carbonate solution. The pH was kept between 10-12 by additionsof 1 N NaOH. The reaction mixture was maintained at a temperature ofabout 30° C. for about 11/2 hours.

The resultant BPO--D--GL conjugate was separated from unreactedpenicillin salt by dialysis against 0.1 M NaHCO₃ containing 1 percentDEAE cellulose. Dialysis was allowed to proceed for a period of aboutone week.

Analysis of the BPO--D--GL conjugate obtained, by the method referred toearlier, indicated that the BPO:D--GL molar ratio was BPO₆₃ --D--GL.

EXAMPLE III Insulin-D--GL Conjugate

A 50 mg portion of porcine insulin was dissolved in 0.01 Methylenediaminetetraacetic acid (EDTA) at a pH of 3.1. The dissolvedinsulin was dialyzed against the same EDTA solution overnight. Thedialyzing medium was changed to 0.033 M borate buffer at a pH of about9.5, containing 2.5×10⁻⁵ M EDTA. Toluene-2,4-diisocyate (TDIC) was addedto the insulin solution at 0° C. The reaction mixture was stirredvigorously at 0° for about 30 min. and then centrifuged at 12,000 g for10 min. at a temperature of about 2° to 4° C. The supernatant wasdecanted into a stoppered test tube and the test tube placed in an icebath. The reaction was allowed to proceed an additional hour at ice bathtemperatures.

A 50 mg portion of D--GL copolymer having an average molecular weight of64,000 and a glutamic acid:lysine molar ratio of 60:40, was dissolved in0.033 M borate buffer (pH 9.5), containing 2.5×10⁻⁵ M EDTA. The pH wasadjusted to about 10-12 with 2 N NaOH.

The D--GL solution was added to the insulin solution; the molar ratio ofinsulin to D--GL added to the reaction mixture was 10:1. The reactionwas allowed to proceed for one hour at about 35° to 40° C. The reactionmixture was then dialyzed against 0.1 M (NH₄)₂ CO₃ containing 2.5×10⁻⁵ MEDTA and fractionated on a Sephadex 675 column using 0.01 M NH₄ HCO₃,containing 2.5×10⁻⁵ M EDTA as the eluant. The conjugate was furtherdialyzed against distilled water and lyophilized.

EXAMPLE IV Nucleotide-D--GL Conjugate

Oligodeoxynucleotide (trimers and/or tetramers) were prepared by DNase 1digestion of calf thymus DNA followed by fractionation on a DEAESephadex A25 column. The procedure involved using a 0-0.4 M LiCl lineargradient in 5 mM tris(hydroxymethyl)aminomethane ("TRIS") -7 M ureabuffer (pH=7.6). Urea was removed from the oligodeoxynucleotidesproduced, by chromatographic techniques, using distilled water as theeluant.

The oligodeoxynucleotide produced is reacted with D--GL copolymer,having a average molecular weight of about 64,000 and a glutamicacid:lysine molar ratio of 60:40. The reaction is conducted in distilledwater, using 1-ethyl-3-diisopropylaminocarbodiimide hydrochloride (EDC)as the coupling agent.

The resultant conjugate is separated from impurities and unreactedstarting material by dialysis for about one week at about 2° to 4° C.[See J. of Immun., 96, 373 (1966)].

Oligoneucleotides were also prepared by DNase 1 digestion of calf thymusDNA followed by passing the material through a filter having a molecularweight cut-off of about 10,000. A suitable filter is available fromAmicon, division of Rohm and Haas Co., Independence Mall West,Philadelphia, Pa., 19105, under the trade designation PM-10. Thefiltrate was used as the source of the oligodeoxynucleotide.

Nucleotides are composed of a heterocyclic base, a five carbon sugar andphosphate. In turn, nucleic acids are polymers composed of fourdifferent nucleotides, linked together by phospho-diester bonds.Oligonucleotides are small polymers, usually obtained from nucleicacids, composed of fewer than 10 nucleotides which are linked togetherby phospho-diester bonds. The diester bonds make the nucleotidesrelatively complex chemical substances. Antibodies specific for nucleicacid are not only specific for the base and/or sugar but also for thephosphate backbone containing these nucleotides. Thus, by using a smalloligonucleotide attached to a nonimmunogenic carrier (e.g., D--GL), anucleic acid-like environment directly related to the pathological stateof an autoimmune disease is obtained. In contrast, prior art inductionof tolerance to nucleosides, which do not contain phospho-diester bonds,is more closely related to hapten--D--GL tolerance, e.g., Dnp--D--GL.Induction of tolerance to oligodeoxynucleotides can be considered to beequivalent to induction of tolerance to nucleic acid.

EXAMPLE V Ragweed Antigen E--D--GL Conjugate

A 50 mg portion of D--GL copolymer having an average molecular weight of64,000 and a glutamic acid:lysine molar ratio of 60:40 was dissolved in2 ml of distilled water. The solution was cooled to 0° C. in an ice bathand stirred. A 50 mg portion of N-ethyl-5-phenylisoxazolium 3'-sulfonate(Woodward's Reagent), was dissolved in 0.5 ml of distilled water, addedto the D--GL solution and stirring continued for about 1 hour at 0° C.The pH was raised to 7.5-8.0 with 2 N NaOH.

A 5 mg portion of ragweed antigen E, available from WorthingtonBiochemicals, Inc., Freehold, N.J., 07728, was dissolved in distilledwater and added to the D--GL-Woodward's Reagent solution. The antigen Ehad a molecular weight of 37,800; nitrogen was 17.1 percent andcarbohydrate was 0.2 percent. The S value was 3.05 and the extinctioncoefficient (280μ) of a 1 percent solution at 1 cm was 11.3.

The mixture was stirred for 24 hours at 6° C. The reaction mixture wasfractionated on a Sephadex G-100 column using 0.01 M NH₄ HCO₃,containing 0.1 M NH₄ HCO₃ as the eluant. The tubes containing the firstpeak were pooled together and the conjugate further dialyzed againstdistilled water and lyophilized.

The present invention has therapeutic implications for treatment ofpathological manifestations involving any antibody dysfunction. Since,as indicated earlier, a large number of individuals are hypersensitiveto environmental antigens, a method of treatment to alleviate theseallergic symptoms would be of immense therapeutic value. By the presentinvention, IgE and IgG antibody production in response to specificsensitizing antigens is greatly suppressed.

Accordingly, the antigen-D-GL conjugate and treatment of the presentinvention includes a wide variety of environmental antigens, denoted asallergens. For example, representative allergens include, but are notlimited to: drugs such as penicillin; hormones such as insulin; pollenssuch as ragweed, bermuda grass, orchard grass and timothy grass, flowerpollen such as Chrysanthemum leucanthemum, and tree pollen such asbirch; venoms such as bee wasp and snake; animal danders such as horsedander; animal epidermis such as cat epithelium; food protein allergenssuch as haddock, strawberries; house dust mite; fungi such as baker'syeast (Saccharomyces cerevisiae); molds such as Alternaria tenuis;toxins such as diphtheria; toxoids such as tetanus; proteins such asovalbumin; enzymes such as trypsin, chymotrypsin and ficin; andderivatives of these allergens.

In addition to the environmental allergens described above, whichproduce allergic symptoms in hypersensitive individuals, the instantinvention has therapeutic value for treatment of autoimmune disease.Autoimmune diseases can affect almost every part of an individual'sbody. Some responses are directed to organ-specific antibodies, and canbe directed to a particular cell type, e.g., parietal cells of gastricmucosa in pernicious anemia. Other responses are directed to widelydistributed antigens and are associated with disseminated diseases,e.g., antinuclear antibodies in systemic lupus erythematosus (SLE). Instill other diseases, the responses are intermediate between theseextremes, e.g., in Goodpasture's disease, characterized by chronicglomerulonephritis and pulmonary hemorrhages, antibodies are depositedon basement membrane of kidney glomeruli and lung parenchyma.

Antibodies are also formed to specific cell receptor sites as occurs inmyasthenia gravis where antibodies to acetylcholine receptor sitesinterfere with the transmission of neural impulses. Antibodies can beformed to insulin receptor sites, blocking the binding of insulin to thecells, which interferes with the regulatory action of the hormone.

Representative autoimmune diseases, and the antigen responsible include:

    ______________________________________                                        Thyroid  Hashimoti's thyroiditis                                                                         Thyroglobulin;                                              (hypothyroidism)  and                                                         Thyrotoxicosis    Thyroid cell                                                (hyperthyroidism) surface;                                           Gastric  Pernicious anemia Intrinsic                                          factor(1)                                                                     mucosa   (vitamin B.sub.12 deficiency)                                                                   Parietal cells                                     Adrenals Addison's disease Adrenal cell                                       Skin     Pemphigus vulgaris                                                                              Epidermal cells                                                               and                                                         Pemphigoid        Basement membrane                                                             between                                                                       epidermis-                                                                    dermis                                             Eye      Sympathetic ophthalmia                                                                          Uvea;                                              Red cells                                                                              Autoimmune hemolytic                                                                            Red cell surface;                                           anemia                                                               Platelets                                                                              Idiopathic thrombocytopenic                                                                     Platelet surface;                                           purpura                                                              Skeletal and                                                                           Myasthenia gravis Muscle cells                                       heart muscle               and thymus                                                                    "myoid" cells;                                     Liver    Primary biliary cirrhosis                                                                       Mitochondria                                       (biliary                   (mainly);                                          tract)                                                                        Salivary and                                                                           Sjogren' s disease                                                                              Includes secretory                                 lacrimal                   ducts, mito-                                       glands                     chondria,                                                                     nuclei and IgC:                                    Synovial Rheumatoid arthritis                                                                            Fc domain of IgG                                   membranes                                                                     etc                                                                           ______________________________________                                    

By the method of the instant invention it appears that alleviation ofautoimmune disease can be achieved by coupling to D--GL the appropriateantigen which has been implicated in the various autoimmune diseasesreferred to above. Suppression of formation of the IgG antibodies to theself-antigens would be of therapeutic value.

What is claimed is:
 1. A therapeutic immunosuppressive agent capable ofinducing specific immunological tolerance to an antigen by suppressionof antibody response, comprising a conjugate of D-glutamic acid:D-lysinecopolymer and the antigen ragweed antigen E.
 2. An immunosuppressiveagent as claimed in claim 1 wherein said copolymer has a molecularweight of from about 34,000 to 64,000 and a glutamic acid:lysine molarratio of about 60:40.
 3. A method of preparing a therapeuticimmunosuppressive agent comprising a conjugate of D-glutamicacid:D-lysine copolymer and an antigen which is an allergen or aself-antigen, comprising the steps of reacting D-glutamic acid:D-lysinecopolymer, having an average molecular weight of from about 34,000 to64,000 and a glutamic acid:lysine molar ratio of about 60:40, with theantigen ragweed antigen E.
 4. A method claimed in claim 3 wherein thereaction mixture is maintained at a pH of about 10-12.